Targeting gene expression to living tissue using jet injection

ABSTRACT

The present invention provides a method of targeting transient gene expression and stable gene expression from the exogenous administration of a DNA sequence, which sequence is less than a complete genome, wherein said DNA sequence encodes RNA and protein, or RNA only, to differentiate tissue of living organisms wherein said DNA sequence through a jet injector technique, and said DNA sequence of less than a complete genome is expressed in a living organism. The present invention further provides a flexible multi-nozzle injector device with a wide surface area to allow molding of the injector nozzle to the surface contours of the tissue. Another aspect of the present invention provides an injection device having a long nozzle for injection of DNA deep into the host tissue. Also, in a further aspect the present invention provides an injector device modified to be used with and/or inject through an endoscopic device.

FIELD OF THE INVENTION

[0001] The present patent application involves the introduction of DNAsequences encoding RNA and proteins into the differentiated tissues of aliving organism. It involves a procedure by which DNA sequences can betargeted to differentiated an undifferentiated tissue and for enhancingthe expression of DNA sequences in specific target cells.

BACKGROUND OF THE INVENTION

[0002] It is known in this field that a full length cottontail rabbitpapillomavirus (CRPV) DNA genome of the Washington B strain of the viruscan be injected into rabbit skin as an episome by several methods (See,Brandsma et al, Proc. Natl. Acad. Sci., U.S.A., Vol. 88, pages 4816-4820(June 1991)). In the Brandsma publication, inter alia, the authorsdiscuss, as one of the several methods of inoculating with the episome,the use of a ped-o-jet (a standard hypodermic jet injector gun) forinjecting the entire viral genome of the cottontail rabbitpapillomavirus (CRPV) DNA into rabbit skin. Prior to injection of thefull length genome Brandsma purifies it either by using standard cesiumchloride centrifugation or by using polyethylene glycol precipitationfollowed by proteinase K digestion. Equivalent purification results wereobtained with DNA purified by either method.

[0003] The Brandsma publication alludes to the possibility that furtherresearch might lead to other forms of DNA that might also be injectedusing the jet injection-technique, but there is no data or descriptionalbasis to lead one to expect any likelihood of success with injecting DNAif less than a complete genome episome.

[0004] The four different methods discussed in Brandsma et al forinoculating rabbit skin involved (a) epithelial scarification with arazor blade followed by smearing on of DNA, (b) scratching the skin withthe back of an 18-gauge needle followed by smearing on of DNA, (c)intradermal inoculation and puncture 200 times with a 27-gauge needle,and (d) interdermal inoculation using a PED-O-Jet injector (StirnIndustries, Dayton, N.J.).

[0005] The four types of inoculations were performed on four to fivepound random-bred New Zealand white female rabbits, anesthetized withketamine hydrochloride (44 milligrams per kilogram) after clipping theirbacks free of hair. The inoculant contained 70 micrograms of supercoiledCRPV-p LAI1 DNA (wild type or mutant) per site in 0.1 milliliter of 0.15M NaCl or 10 mM Tris.HCl/1 mM EDTA.

[0006] Inoculation methods (a) and (b) were not very efficient, but theintradermal inoculation and puncture (manual) and the jet injectormethods both induced papillomas. The intradermal manual method inducedpapillomas in 15-25% of the inoculated sites on all the rabbits. Thejet-injector caused 23-81% of the inoculated sites to form papillomas.

[0007] The Brandsma paper concluded that there was some advantage as tothe total number of papillomas produced and a savings of time for theinoculation. However, it was not clear whether the increased rate ofproducing papillomas from the DNA episomes could also be accounted forpossibly of different depths at which the injection was done into thetissue using the manual or the jet method. Further, there is no data forthe manual method using a larger diameter (smaller gauge) needle for theinjection. The 27 gauge needle used has a very small diameter and couldpossibly have broken the episome DNA into fragments.

[0008] In other words, Brandsma is not clear as to whether the jetinjector and the manual injection were done at the same depth in thetissue. Possibly, one of the two methods injected deeper into the tissueor injected over a wider area of tissue. Specifically a different manualinjection technique (such as injecting while withdrawing the needleinstead of at just one depth) may have been much more successful thanthe technique used by Brandsma. Thus, the data produced in Brandsma isinconclusive on this point.

[0009] Moreover, in Brandsma there is no experimental evidence or datathat would provide any reasonable basis for believing that DNA fragmentswhich are not capable of producing an episome would be expressed if theywere injected either manually or using the jet injection.

[0010] Accordingly, there is a need in this art for an improved methodfor initiating gene expression for DNA which corresponds to less than acomplete genome, i.e., gene fragments, in a host and for improvedequipment with which to perform these injections.

SUMMARY OF THE INVENTION

[0011] It is an object of the present invention to provide a method oftargeting transient gene expression and stable gene expression from theexogenous administration of a DNA sequence, which sequence is less thana complete genome, wherein said DNA sequence encodes RNA and protein, orRNA only, to differentiate tissue of living organisms wherein said DNAsequence through a jet injector technique, and said DNA sequence of lessthan a complete genome is expressed in a living organism.

[0012] Another object of the present invention is to provide a flexiblemulti-nozzle injector device with a wide surface area to allow moldingof the injector nozzle to the surface contours of the tissue.

[0013] Still another object of the present invention is to provide aninjection device having a long nozzle for injection of DNA deep into thehost tissue.

[0014] It is a further object of the present invention to provide aninjector device modified to be used with and/or inject through anendoscopic device.

BRIEF DESCRIPTION OF THE DRAWING

[0015]FIG. 1 is a diagram showing an improved jet injection device forinjection of DNA into the human female cervix.

[0016]FIG. 2 is a diagram showing an improved jet injection device forinjection of DNA into living tissue using an endoscopic device.

[0017]FIG. 3 is a diagram showing an improved jet injection nozzle forthe deep injection of DNA into tissues.

DETAILED DESCRIPTION OF THE INVENTION

[0018] The present invention provides a method of targeting transientgene expression and stable gene expression from the exogenousadministration of a DNA sequence of less than a complete genome, whereinsaid DNA sequence encodes RNA and protein, or RNA only, to differentiatetissue of living organisms, wherein said DNA sequence is administeredthrough a jet injector technique and said sequence of less than acomplete genome is expressed in a living organism.

[0019] In another aspect the present invention relates to a flexiblemulti-nozzle injector with a wide surface area to allow for molding ofthe injector nozzle through the surface of the tissue. In another aspectof the invention, the invention relates to a injector with a longinjection port for injecting deep into tissue in a regulated fashion. Astill further aspect of the present invention is a injector with aninjector nozzle having an injector port which has been modified to usewith an endoscope.

[0020] The improved jet injection device for injection of the humanfemale cervix consists of a jet injection nozzle which is small enoughto position on the human female cervix (which is located within thevagina). The nozzle is located at the end of a length of injectiontubing sufficient to position the injection nozzle on the surface of thecervix, with the jet propulsion system located outside the body. Thedevice is held in position at the cervix by cervical clamps. The nozzleis moved over the entire surface of the cervix by moving a platformwhich is located outside the body. The movements of the platform arecontrolled by computer to insure that small movements will be made(approximately four millimeter is optimal) and the entire cervicalsurface is covered with injections. As an alternative, a manually drivendevice can be substituted for the computer.

[0021] The improved jet injection device for injection of living tissueusing an endoscopic device may be used in endoscopy, bronchoscopy,cystoscopy, colonoscopy, laparoscope, or other similar procedures usinga type of endoscopic device. The endoscopic injection device is coupledto conventional endoscopic technology. The endoscope is used tovisualize the tissue to be injected and to position the jet injectiondevice at that sight. The jet injection nozzle and tubing is made smallenough to enter the appropriate body orifice for each individualendoscopic device.

[0022] The improved jet injection nozzle for deep injection of tissueare designed to be able to deliver a jet injection to a tissueinternally. The needle injection device is inserted into a tissue at therequired depth. The injection is then performed. Two types of needleinjections can be used. A first type of device delivers the injectionthrough a single port. A second type of device delivers the injectionthrough multiple ports, i.e. two or more injection ports. The diagram inFIG. 3 shows one port at the end of the needle and one port on eachside. Additional ports may be added as needed for particular tissues thediameter of the needle is varied for each type of tissue, the range isusually between 12 and 18 gauge, which is a large diameter needle.

[0023] The method according to the invention preferably involves thesteps of a member selected from the group consisting of (a) ablation ofmalignant cells, (b) ablation of cells infected with specific viruses,(c) gene therapy, (d) immunization, (e) generation of transgenicorganisms, (f) converting secretory cells of living organisms intobio-reactors for producing a protein, (g) modifying the expression ofindigenous gene, (h) providing a means for studying the effects ofspecific proteins in differentiated and undifferentiated tissue, (i)generating an animal model system for human diseases, and (j) inducingwound healing via the production of specific growth factor genes.

[0024] Further preferred is a method wherein the secretory cells of theliving organism are mammary or bladder cells.

[0025] Another preferred aspect of the invention is a method accordingto wherein the exogenous DNA sequence is a DNA sequence selected fromthe group consisting of a DNA sequence having enhancer/promotor andother regulatory elements, a DNA sequence which can be transcribed intoan RNA which RNA can be (a) translated into a protein, (b) includes atranscriptional termination signal, and (c) may encode a signal peptidewhich allows a protein to be exported from the cell, a DNA sequencewhich targets a gene for incorporation into the genome, a DNA sequencewhich directly replicates in eukaryotic cells, and a plasmic sequencewhich allows DNA replication in prokaryotic cells.

[0026] The preferred exogenous DNA sequences are DNA sequences whichnaturally occur in a genome but are not a complete genome, or a DNAsequence which is constructed using enhancer/promoter components,termination signals, and may include a signal peptide coding sequencesfrom different genes which are combined to directly express in aspecific manner. Even more preferred is a method wherein theenhancer/promotor sequence is a naturally occurring element such as theHCMVIE1 promotor/enhancer, or the enhancer/promoter sequencesconstructed using specific DNA elements which mediate binding byspecific transcription factors to directly express only in specific celltypes. A preferred synthetic promoter is composed of a generic TATA boxand binding cites for the E2 transcription factor and coded by thepapillomavirus genome, wherein the gene expression from such a promotoris confined to cells expressing the E2 protein from papillomavirus.

[0027] The development of techniques to transfect DNA into somatictissues is motivated by the potential value of this technology for genetherapy; (Friedmann, T. (1989) Science 244, 1275-1281) and for geneticimmunization; (Tang, D., DeVit, M., and Johnston, S. A. (1992) Nature356, 152-154). Techniques that have been described include needleinjection of DNA in a 5% sucrose solution; (Wolff, J. A., Malone, R. W.,Williams, Chong, W., Acsadi, G., Jani, A., and Feigner, P. L. (1990)Science 247, 1465-1468) or following co-precipitation with CaCl₁₂;(Benvenisty, N., and Reshef, L. (1986) Proc. Natl. Acad. Sci. USA 81,5849-5852; Nabel, E. G., Plautz, G., and Nabel, G. J. (1990) Science249, 1285-1288; and Seeger, C., Ganem, D., and Varmus, H. E. (1984)Proc. Natl. Acad. Sci. USA 81, 5849-5852) encased in liposomes; (Nabel,E. G., Plautz, G., and Nabel, G. J. (1990) Science 249, 1285-1288 andNicolau, C., Le Pape, A., Soriano, P., Fargette, F., and Juhel, M-F 1983Proc. Natl. Acad. Sci. USA 80, 1068-1072) in combination with nuclearproteins; (Kenaeda, Y., Iwai, K., and Uchida, T. (1989) Science 243,375-378) or another carrier; (Wu, G. Y., and Wu, C. H. (1988) J. Biol.Chem. 263, 14621-14624). Somatic cells have also been transformed byparticle bombardment; (Zelenin, A. V., Alimov, A. A., Titomirov, A. V.,Kazansky, A. V. et al (1991) FEBS Lett. 280, 94-96; Yang, N. -S.,Burkholder, J., Roberts, B., Martinell, B., and McCabe, D. (1990) Proc.Natl. Acad. Sci. USA 87, 9568-9572 and Klein, T. M., Arentzen, R.,Lewis, P. A., and Fitzpatrick-McElligott, S. (1992) BIO/TECHNOLOGY 10,286-291) or using retroviral vectors; (Friedmann, T. (1989) Science 244,1275-1281). The entire cottontail rabbit papillomavirus (CRPV) genomehas been jet injected by an air pressure propulsion system into rabbitepithelium as discussed above; (Brandsma, J., Yang, Z. -H., Barthold, S.W., and Johnson, E. A. (1991) Proc. Natl. Acad. Sci. USA 88, 4816-4820).

[0028] Surprisingly, the present Applicants have found thatcorresponding genes to less than a complete genome can be expressed byjet injection of reporter genes through the skin surface to transfectskin, muscle, fat, and mammary tissue of living animals. Further,Applicants have discovered that the isolated mouse mammary gland can besuccessfully transfected by this technique.

[0029] The present invention will be more clear by the followingspecific application of its concepts.

Expression Vectors

[0030] Three hybrid genes were used. The first contained humancytomegalovirus immediate early gene 1 (HCMVIE1) enhancer/promotersequences and the bacterial chloramphenicol acetyl transferase (CAT)gene. This hybrid gene is expressed in at least 28 different mousetissues and it is also active in a wide variety of tissue culture celllines; (Furth, P. A., Hennighausen, L., Baker, C., Beatty, B., andWoychik, R. (1991) Nuc. Acids Res. 19, 6205-6208). The second vectorcontained whey acidic protein (WAP) promoter sequences between −450 and+24 and the CAT gene. In contrast to the universally expressed HCMVIE1enhancer/promoter, the WAP promoter is active only in differentiatedmammary tissue; (Pittius, C., Hennighausen, L., Lee, E., Westphal, H.,Nicoli, E., Vitale, J., and Gordon, K. (1988) Proc. Natl. Acad. Sci. USA85, 5874-587816). The third expression vector contained HCMVIE1enhancer/promoter sequences and the bacterial β-galactosidase gene(CLONTECH Laboratories, Inc., Palo Alto, Calif.).

Animals

[0031] Four male and 15 female C57B6/SJL mice between 15 to 24 weeks ofage were injected with gene constructs in this study. Females were matedand injected 10 to 15 days after copulation. Pregnancy was confirmed atthe time of autopsy. Mammary glands of four Rambouillet ewes, near theend of lactation (5 to 7 weeks after parturition) were also injected.All animals were observed for morbidity from jet injection. The injectedtissues were examined for evidence of tissue damage at the time ofautopsy or biopsy.

Jet Injection of DNA

[0032] Supercoiled DNA fragments at 1 μg/μl in 1 mM TRIS-0.1 mM EDTAwere introduced into specific tissues with a Ped-O Jet injection (StirnIndustries, Dayton, N.J.). Each mouse was anesthetized with 0.7 ml0.175% 2,2,2,-tribromoethanol in 3.5% 30 amyl alcohol (Avertin) and thesite(s) of injection were lightly shaved with a razor.

[0033] The injector was placed on the skin surface and fired. Betweenone and three injections were made at the surface of the skin overlyingthe inguinal mammary glands and the thigh muscle. One to threeinjections in volumes of 100 μl or 300 μl per injection were made ateach site. In some cases the inguinal mammary glands were removedimmediately after injection and cultured.

[0034] In other experiments the inguinal mammary glands were firstexcised and placed in a petri dish, injected and subsequently cultured.The injector was placed at or just above the surface of the mammaryglands and each gland was injected five times with a volume of 100 μl.

[0035] Because it was not known if jet injected solutions wouldpenetrate the skin overlying sheep mammary glands, three sites ofinjection were tested during the initial experiment in sheep. Injectionswere performed on the skin surface, into subcutaneous fat and at thesurface of the fascia surrounding the mammary gland. Between one andthree injections were made at each site. Volumes of the injection rangedfrom 100 μl to 500 μl. In subsequent sheep experiments injections wereperformed at the skin surface. In two cases biopsies of the sheep glandwere removed immediately after injection and cultured.

Mammary Gland Explant Cultures

[0036] Mammary tissue was transferred into medium M-199 containing 100U/ml penicillin, 100 μg/ml streptomycin and 0.25 μg/ml Fungizone and 1μg/ml insulin. Explant cultures were prepared as previously described;(Shamay, A., Zeelon, E., Ghez, Z., Cohen, N., Mackinlay, A. G. andGertler, A. (1987) J Endocrinol. 113, 81-88) and cultured at 37° C. inmedium supplemented with insulin (1 μg/ml), cortisol 0.5 μg/ml), andprolactin (1 μg/ml). The media was changed every 24 hours.

Analysis of Chloramphenicol Acetyl Transferase (CATS Activity

[0037] Mice were killed by cervical dislocation 48 hours afterinjection, and targeted tissues were removed. Cultured mouse and sheepmammary glands were harvested after 48 hours in culture. In two casesthe injected sheep tissues were harvested 24 or 48 hours after injectionby excisional biopsy following local lidocaine anesthesia. In threecases the sheep tissues were harvested 48 hours after injection duringautopsy. The three sheep were killed by stunning them with a captivebolt followed by exsanguination. Protein extracts were prepared,concentration of protein determined and CAT assays performed aspreviously described; (Leonard, J. M., Khillan, J. S., Gendelman, H. E.,Adachi, A., Lorenzo, S. J., Westphal, H., Martin, M., and Meltzer, M. S.(1989) AIDS Res. Hum. Retroviruses 5, 421-430). The acetylated andnonacetylated forms of chloramphenicol were quantitated by radioanalyticimagining (AMBIS) and degree of acetylation was calculated. Thepicograms of CAT enzyme present in each tissue sample was calculated bycomparison of experimental values to a known standard (5 Prime 3 Prime,West Chester, Pa.).

Analysis of β-galactosidase Activity

[0038] Injected tissues were fixed in paraformaldehyde prior toincubation in an X-Gal staining solution (19). Tissues were sectioned,counterstained with neutral fast red and examined under the microscopefor evidence of β-galactosidase activity.

RESULTS CAT Expression in Living Animals

[0039] CAT activity was measured in skin, muscle, fat, and mammarytissue following jet injection of the HCMVIE1 CAT expression vectors atthe skin surface (Table 1). In the mouse, activity was detected muscletissue 2-3 mm distant from the site of injection, the skin surface. Inthe sheep, mammary gland cells 1 to 3 cm distant from the site ofinjection, the skin, were successfully transfected (Table 1). CATactivity could not be detected in all samples, most likely secondary toa combination of variability in transfection efficiency and samplingerror.

CAT expression in Cultured Mammary Glands

[0040] The HCMVIE1 CAT expression vector was active following jetinjection into both mouse and sheep mammary gland cultured in vitro. TheWAP CAT vector was expressed in the mouse mammary gland but was nottested in the sheep mammary gland (Table 1).

β-galactosidase Expression in Living Animals

[0041] β-galactosidase activity was detected in mouse skin and mammarygland following jet injection of a β-galactosidase expression vector atthe skin surface.

Morbidity and Mortality from Jet Injection

[0042] There was no significant morbidity in the sheep following jetinjection and they continued to lactate after the procedure. Resolvinghematoma were found at 2 out of 6 cites of injection at the time ofautopsy. The only morbidity in the male mice was occasional transientbleeding. No evidence of injury was found at the time of autopsy 48hours later.

[0043] There were 4 mortalities out of the 15 pregnant mice injectedinto the skin overlying the mammary gland. Three immediately hemorrhagedextensively following injection and were killed by cervical dislocation.One mouse died 36 hours following injection. An extensive abdominalhematoma was found at autopsy.

[0044] All of the remaining mice appeared to be healthy during the 48hours following injection. None had evidence of hemorrhage or extensivetissue damage at the time of autopsy. A 0.5 mm diameter path ofinjection through the skin, mammary gland and peritoneum could be tracedin two of the mice.

[0045] Table 1 below illustrates the results of the above tests. TABLE 1CAT Samples enzyme, range Gene Total Number CAT Species Construct Tissuenumber positive (pg/mg protein) Mice HOMVIE1 Skin 3 1   2.2 Muscle 3 3 1.0-8.2 Mammary 3 2  1.4-1.9 gland Mammary 3 3 43.6-256.0 gland cultureWAP-CAT Mammary 1 0 <0.5 gland Mammary 2 2 39.8-43.6 gland culture SheepHOMVIE1 Skin 7 2  0.6-2.0 Fat 7 3  2.4-228.0 Mammary 7 4  0.6-185.0gland Mammary 2 2  0.7-1.5 gland culture

[0046] As is clear from the above described experiments, their resultsdemonstrate that differentiated tissues of living animals and isolatedorgans can be transfected with DNA using a jet injection technique. Theuse of jet injection for introducing DNA into somatic tissues may haveadvantages over transfection by particle bombardment. First, no metalneeds to be introduced with the DNA and second, jet injection can beused to transfect cells millimeters to centimeters beneath the skinsurface while particle bombardment is reported to be limited to thefirst 8-20 cell layers; (Klein, T. M., Arentzen, R., Lewis, P. A., andFitzpatrick-McElligott, S. (1992) BIO/TECHNOLOGY 10, 286-291).

[0047] Needle injection of DNA into tissue in either a sucrose orlipofection solution are alternative methods. However, we were unable todetect CAT activity in sheep mammary gland following needle injection ofDNA in TE (data not shown).

[0048] Suspending gene constructs in solutions which enhance cellularuptake of DNA may improve the efficiency of jet injection.

[0049] Important features of jet injection are the ability to injectthrough intact skin, and the minimal morbidity and pain associated witheach injection. These features would permit the practical use ofmultiple injections to improve transfection efficiency. Jet injectioncan be used to transiently express genes controlled by differentiationspecific factors. The WAP promoter is inactive in virtually all cellculture lines; (Doppler, W., Villunger, A., Jennewein, P., Brduscha, K.,Groner, B., and Ball, R. K. (1991) Mol. Endocrinol. 5, 1624-1632) andthe use of transgenic animals has been the predominant means to studythe activity of the promoter; (Hennighausen, L. (1992) J. Cell.Biochem., in press). Jet injection may offer a less costly andtime-consuming method to analyze regulatory elements. The same approachmay be applied to other tissues as well.

[0050] In summary, DNA can be introduced into differentiated somaticcells by jet injection. Modifications to the technique may improvetransfection efficiency. The use of replicating vectors could increaseexpression levels and enhance integration of the expression vector;(Niwa, H., Yamamura, K., and Mizazaki, J. (1991) Gene 108, 193-200). Thetechnique may be useful for genetic immunization, to deliver somaticgene replacement treatment and to target gene therapy to tumor cellsusing toxin or apoptosis genes.

DETAILED DESCRIPTION OF THE DRAWINGS FIG. 1

[0051]FIG. 1 is a diagram showing an improved jet injection device forinjection of DNA into the human female cervix. As shown in FIG. 1, thisimproved jet injection device for injection of the human female cervixconsists of a jet injection nozzle which is small enough to position onthe human female cervix (which is located within the vagina). FIG. 1shows a diagram of the vagina at the location of the cervix, the cervix,the injection device, the supporting system for the injection device,and a cervical clamp or clamps holding the injection device in positionin the vagina.

[0052] The injection device is shown by FIG. 1 to have an injectionnozzle contacting the cervix, the injection tubing connecting theinjection nozzle and the jet propulsion system, the jet propulsionsystem, a fixed platform for positioning the nozzle, a computer nozzleis located at the end of a length of injection tubing sufficient toposition the injection nozzle on the surface of the cervix, with the jetpropulsion system located outside the body. The jet propulsion system issimilar to that of the well-known Ped-O-Jet system, which is readilyadapted to fit the remainder of the device which is shown in FIG. 1. Itis connected to the injection nozzle portion by injection tubing.

[0053] The device nozzle, as shown in FIG. 1, is held in position at thecervix by cervical clamps. The nozzle is moved over the entire surfaceof the cervix by moving a platform which is located outside the body,which platform guides the nozzle. The movements of the platform arecontrolled by computer to insure that small movements will be made(approximately four millimeter movements are optimal) and the entirecervical surface is covered with injections. As an alternative, amanually driven device can be substituted for the computer.

[0054] The dimensions for the device and its components as shown in FIG.1 are for illustration purposes only and can be readily adapted to otheracceptable parameters by those having routine skill by us of routineexperimentation to optimize the device in a particular instance.

FIG. 2

[0055]FIG. 2 is a diagram showing an improved jet injection device forinjection of DNA into living tissue using an endoscopic device. ThisFigure shows an endoscopic device with its controls, an injectionnozzle, a jet propulsion system (generally as described in thediscussion of FIG. 1, above), and an injection tubing apparatusconnecting the injection nozzle with the jet propulsion system. ThisFigure shows the injection device system separate from the endoscopicdevice, but combining the two pieces of apparatus together in a singledevice is an optional embodiment of the present invention.

[0056] The improved jet injection device for injection of living tissueusing an endoscopic device may be used in endoscopy, bronchoscopy,cystoscopy, colonoscopy, laparoscope, or other similar procedures usinga type of endoscopic device. It works generally in the same manner asdiscussed in the description of FIG. 1, above, but the endoscopic deviceis used to view the injection site and aid in the position of theinjection nozzle portion of the injection device.

[0057] The endoscopic injection device is coupled to conventionalendoscopic technology. The endoscope is used to visualize the tissue tobe injected and to position the jet injection device at that sight. Thejet injection nozzle and tubing is made small enough to enter theappropriate body orifice for each individual endoscopic device.

[0058] The dimensions for the device and its components as shown in FIG.2 are for illustration purposes only and can be readily adapted to otheracceptable parameters by those having routine skill by us of routineexperimentation to optimize the device in a particular instance.

FIG. 3

[0059]FIG. 3 is a diagram showing an improved jet injection nozzle forthe deep injection of DNA into tissues. FIG. 3 shows two embodiments ofthe deep injection nozzle. As shown in this Figure the injection nozzlemay have one or more ports on the nozzle, which are located at the tip,along the transverse axis of the nozzle, or both at the tip and alongthe transverse axis of the nozzle. Injection device 1 of FIG. 3 shows asingle deep tissue injection nozzle, whereas device 2 of FIG. 3 showsmultiple deep injection nozzles (e.q., two injection nozzles). More thantwo deep injection nozzles may be used with device 2, if required in aparticular instance to obtain optimal results. The propulsion system andtubing connecting the nozzle (or nozzles) with the propulsion system asessentially as described generally in FIG. 3.

[0060] The improved jet injection nozzles of FIG. 3, for deep injectionof tissue, are designed to be able to deliver a jet injection to atissue internally. The needle injection device is inserted into a tissueat the required depth. The injection is then performed. Two types ofneedle injections can be used. Optionally the deep injection device canalso have a computer guided or manually guided means to set the exactdepth at which the nozzle is inserted into the subject tissue.

[0061] As shown in FIG. 3, and discussed above a first type of devicedelivers the injection through a single port (device 1), and a secondtype of device delivers the injection through multiple ports, i.e. twoor more injection ports, (device 2). The diagram in FIG. 3 shows oneport at the end of the needle and one port on each side on the multipleport injection nozzle. Additional ports may be added as needed forparticular tissues to inject at a single level in the tissue or atmultiple levels throughout the tissue as required for optimal results.

[0062] The diameter of each of the needle-like deep injection devices,as shown in FIG. 3, is varied for each type of tissue to obtain optimalresults, the range is usually between 12 and 18 gauge, which is a largediameter needle. An optimal range for the diameter is readily determinedby one of routine skill using only routine experimentation until optimalresults are obtained.

[0063] The dimensions for each of the devices and its respectivecomponents, as shown in FIG. 3, are for illustration purposes only andcan be readily adapted to other acceptable parameters by those havingroutine skill by us of routine experimentation to optimize the device ina particular instance.

[0064] Moreover, the concepts shown in each of FIGS. 1 through 3 may becombined with each other or with the concepts described in other devicesdescribed in this application. Adaptations can be readily made to obtainoptimal results in a particular situation.

[0065] The foregoing description of the specific embodiments will sofully reveal the general nature of the invention that others can, byapplying current knowledge, readily modify and/or adapt for variousapplications such specific embodiments without departing from thegeneric concept and therefore such adaptations are intended to becomprehended within the meaning and range of equivalents of thedisclosed embodiments. It is to be understood that the phraseology orterminology employed herein is for the purpose of description only andnot of limitation.

What is claimed is:
 1. A method of targeting transient gene expressionand stable gene expression in somatic cell tissue by exogenouslyadministering a plasmid expression vector to differentiated somatic celltissue selected from the group consisting of skin, muscle, fat andmammary tissue of living organisms, through a jet injector technique,wherein said plasmid expression vector is expressed in a livingorganism.
 2. A method according to claim 1 further involving the stepsof a member selected from the group consisting of (a) ablation ofmalignant cells, (b) ablation of cells infected with specific viruses,(c) gene therapy, (d) immunization, (e) generation of transgenicorganisms, (f) converting secretory cells of living organisms intobio-reactors for producing a protein, (g) modifying the expression ofendogenous gene, (h) providing a means for studying the effects ofspecific proteins in differentiated and undifferentiated tissue, (i)generating an animal model system for human diseases, and (j) inducingwound healing via the production of specific growth factor genes.
 3. Amethod according to claim 2 wherein the secretory cells of the livingorganism are mammary or bladder cells.
 4. A method according to claim 1wherein said plasmid expression vector comprises DNA sequences selectedform the group consisting of a DNA sequence claiming enhancer/promotorand other regulatory elements, a DNA sequence which can be transcribedinto an RNA which RNA can be (a) translated into a protein, (b) includesa transcriptional termination signal, and (c) may include codingsequences for a signal peptide which allows a protein to be exportedfrom the cell, a DNA sequence which targets a gene for incorporationinto the genome, a DNA sequence which directly replicates in eukaryoticcells, and a plasmid sequence which allows DNA replication inprokaryotic cells.
 5. A method according to claim 4 wherein said DNAsequence is constructed using enhancer/promoter components, terminationsignals, and signal peptide coating sequences from different genes whichare combined to directly express in a specific manner.
 6. A methodaccording to claim 2 wherein the enhancer/promoter sequence is anaturally occurring element such as the HCMVIE1 promoter/enhancer, orthe enhancer/promoter sequences constructed using specific DNA elementswhich mediate binding by specific transcription factors to directlyexpress only in specific cell types.
 7. A method according to claim 4wherein the enhancer/promoter is composed of a generic TATA box andbinding cites for the E2 transcription factor and said enhancer/promoteris coded by the papillomavirus genome, wherein said enhancer/promoter isexpressed in cells capable of expressing the E2 protein frompapillomavirus.
 8. The method of claim 1, wherein said differentiatedtissue is selected from the group consisting of muscle, fat and mammarytissue.
 9. The method of claim 1, wherein said plasmid expression vectorcomprises a promoter-enhancer sequence selected from the groupconsisting of human cytomegalovirus immediate early gene 1 and wheyacidic protein promoter sequence.
 10. The method of claim 1, whereinsaid plasmid expression vector comprises a hybrid gene selected from thegroup consisting of human cytomegalovirus immediate early gene 1 andchloramphenicol acetyl transferase gene; whey acidic protein promotersequence and chloramphenicol acetyl transferase gene; and humancytomegalovirus, immediate early gene 1 and β-galactosidase gene. 11.The method of claim 1, wherein said plasmid expression vector isexpressed in a living organism at about 1 to about 3 cm. Distant fromthe site of injection.
 12. The method of claim 1, wherein said plasmidexpression vector comprises supercoiled DNA fragments of 1microgram/microliter in 1 mM TRIS- 0.1 mM EDTA and is administered involumes between 100 microliters and 500 microliters per injection. 13.An apparatus for jet injection of DNA into cell tissue, comprising: (a)at least one injection nozzle having at least one injection port; (b)injection tubing connecting said injecting nozzle to a jet propulsionsystem affixed to a platform for positioning said injection nozzle; (c)computer means electrically connected to said platform, wherein saidcomputer means is adapted to provide movement to said platform toposition said injection nozzle on the surface of the tissue to beinjected.
 14. An apparatus for jet injection according to claim 8,wherein said injection port is located at the end of said injectionnozzle.
 15. An apparatus for jet injection according to claim 8, whereinsaid injection port is located on the side of said injection nozzle. 16.An apparatus for jet injection according to claim 8, wherein saidplatform comprises a stationary platform connected to a moveableplatform.
 17. An apparatus for jet injection according to claim 8,further comprising an endoscopic device running parallel to saidinjection tubing, wherein said endoscopic device is controlled by anendoscopic control.
 18. An apparatus for jet injection according toclaim 8, said apparatus is adapted for injection of DNA into the humanfemale cervix.
 19. A method of targeting transient gene expression andstable gene expression in mammary tissue by exogenously administering aplasmid expression vector, to mammary tissue of living organisms,through a jet injector technique, wherein said plasmid expression vectoris expressed in a living organism.
 20. A method according to claim 1,wherein said living organism is immunized by said plasmid expressionvector which is expressed in said living organism.
 21. A method ofimmunization comprising the steps of jet injecting an effective amountof a plasmid expression vector, to transform differentiated somatic celltissue of living organisms selected from the group consisting of skin,muscle, fat and mammary tissue, wherein said plasmid expression vectoris expressed in a living organism, and wherein DNA expressed from saidplasmid expression vector immunizes said living organism.